Acylated uridine and cytidine and uses thereof

ABSTRACT

The invention relates to compositions comprising acyl derivatives of cytidine and uridine. The invention also relates to methods of treating hepatopathies, diabetes, heart disease, cerebrovascular disorders, Parkinson&#39;s disease, infant respiratory distress syndrome and for enhancement of phospholipid biosynthesis comprising administering the acyl derivatives of the invention to an animal.

FIELD OF THE INVENTION

[0001] This invention relates generally to acyl derivatives of cytidineand uridine and to the use of those derivatives to deliver exogenousribonucleosides to animal tissue. More specifically, this inventionrelates to the acyl derivatives of cytidine and uridine, and the uses ofthose novel derivatives to deliver these ribonucleosides to animaltissue and thereby to support cellular metabolic functions. Even morespecifically, this invention relates to the use of the novel acylderivatives to treat or prevent a variety of physiological andpathological conditions, including treatment of liver disease or damage,cerebrovascular disorders, respiratory distress syndromes, cardiacdamage, and other clinical conditions.

BACKGROUND OF THE INVENTION

[0002] There are many physiological and pathological conditions ofanimal tissue where the supply of exogenous ribonucleosides may haveuseful therapeutic applications. In a number of physiological andpathological conditions, the administration to an animal of RNA,nucleotides, or individual or mixtures of nucleosides, has been shown toimprove the natural repair processes of the affected cells.

[0003] There are many important metabolic reactions that are usuallyfunctionally subsaturated and limited by availability of eithersubstrates or cofactors. Such rate-limiting compounds may be eithernutritionally essential or synthesized de novo in the body. Underconditions of tissue trauma, infection or adaptation to physiologicaldemand, particularly when cellular repair or regeneration processes areactivated, the optimum nutritional, biochemical, or hormonal environmentfor promoting such repair may be quite different from the requirementsfor normal cell and tissue function. In such cases, therapeutic benefitmay be derived by providing appropriate conditionally essentialnutrients, such as ribonucleosides or metabolites which may be requiredin quantities not usually available from a normal diet. The therapeuticpotential for this strategy of directly supporting the metabolicfunction of damaged or diseased tissues has not been realized incontemporary medical practice.

[0004] At the cellular level of organization, there are specificmetabolic responses to trauma that are involved, in a variety oftissues, in the processes of tissue repair, regeneration, or adaptationto altered functional demand. Most processes of tissue damage and repairare accompanied by a substantial increase in the activity of the hexosemonophosphate pathway of glucose metabolism.

[0005] The hexose monophosphate pathway is the route of formation forthe pentose sugars (e.g., ribose) which are necessary for nucleotide andnucleic acid synthesis. The availability of ribose is rate limiting fornucleotide synthesis under most physiological or pathologicalconditions. Rapid production of nucleotides for the synthesis of nucleicacids and nucleotide-derived cofactors (such as cytidinedi-phosphocholine (CDP choline) or uridine di-phosphoglucose (UDPG)) isessential for the processes of tissue repair and cellular proliferation.Even though nucleotides are synthesized de novo from simpler nutrients,so that there is not an absolute dietary requirement for directnucleotide precursors, many tissues may not have optimal capacity fornucleotide synthesis particularly during tissue repair or cellularproliferation.

[0006] It is possible to bypass the limited capacity of the hexosemonophosphate pathway by providing preformed ribonucleosides directly totissues where they are incorporated in the nucleotide pools via the“salvage” pathways of nucleotide synthesis. It is also possible thatpyrimidine ribonucleosides may exert therapeutic influences throughmechanisms unrelated to the support of nucleotide biosynthesis.

[0007] The effects of the administration of pyrimidine nucleosides, andin particular, uridine and cytidine, on a variety of physiological andpathological conditions in experimental animals, isolated tissues, andto some extent, in humans, have been extensively studied. These aresummarized below.

(1) Heart

[0008] In isolated rat hearts subjected to low-flow ischemia,reperfusion with uridine induced restoration of myocardial ATP levels,total adenine nucleotide content, uridine nucleotide levels, andglycogen content. Ischemia was reported to produce a breakdown ofcreatinine phosphate, ATP, uridine nucleotides and glycogen. Aussedat,J., Cardiovasc. Res. 17:145-151 (1983).

[0009] In a related study, perfusion of isolated rat hearts with uridineresulted in a concentration-dependent elevation of myocardial uracilnucleotide content. Following low-flow ischemia, the rate ofincorporation of uridine was increased twofold. Aussedat, J., et al.,Mol. Physiol. 6:247-256 (1984).

[0010] In another study, isoproterenol was administered to rats whichdepleted cardiac glycogen stores and reduced myocardial UTP andUDP-glucose levels. Despite the spontaneous restoration of myocardialUTP levels, UDP-glucose concentrations remained depressed unless uridineor ribose were administered. Prolonged intravenous infusion of ribose-oruridine resulted in a restoration of myocardial glycogen. Thus, theremay be compartmentation of uridine nucleotides in the heart, with thepools being fed differentially by the salvage or de novo pathways ofpyrimidine synthesis. Aussedat, J., et al., J. Physiol. 78:331-336(1982).

[0011] The effects of nucleosides on acute left ventricular failure inisolated dog heart was studied by Buckley, N. M., et al., Circ. Res7:847-867 (1959). Left ventricular failure was induced in isolated doghearts by increasing aorta pressure. In this model, guanosine, inosine,uridine and thymidine were found to be positive inotropic agents, whilecytidine and adenosine were negatively inotropic.

[0012] Sodium uridine monophosphate (UMP) and potassium orotate werefound to increase the animal's resistance to subsequentadrenaline-induced myocardial necrosis. These compounds reducedmortality and improved myocardial function as assessed by ECG readings,biochemical findings, and relative heart weight. Intravenousadministration of UMP exerted a more pronounced prophylactic effect thandid potassium orotate. Kuznetsova, L. V., et al., Farmakol.-Toksikol2:170-173 (1981).

[0013] In a study on the effects of hypoxia in isolated rabbit hearts,myocardial performance declined while glucose uptake with glycolysis,glycogenolysis and breakdown of adenine nucleotides were reportedlyincreased. Administration of uridine increased myocardial performance,glucose uptake and glycolysis and also diminished the disappearance ofglycogen and adenine nucleotides from hypoxic hearts. Uridine alsoincreased glucose uptake, glycolysis, levels of ATP and glycogen, aswell as myocardial performance in propranolol-treated hearts. Kypson,J., et al., J. Mol. Cell. Cardiol. 10:545-565 (1978).

[0014] In a study of pyrimidine nucleotide synthesis from exogenouscytidine in the isolated rat heart, myocardial cytosine nucleotidelevels were significantly increased by a 30 minute supply of cytidine.Most of the cytidine was recovered as part of cytosine nucleotides anduracil nucleotides. Very little of the cytidine that was taken up wasconverted into uridine nucleotides. These results suggest that theuptake of cytidine can play an important part in myocardial cytosinenucleotide metabolism. Lortet, S., et al., Basic Res. Cardiol.81:303-310 (1986).

[0015] In another study, myocardial fatigue was produced by repeated,brief ligations of the ascending aorta. Intravenous administration of amixture of uridine and inosine after the fifth such ligation temporarilystopped the development of fatigue in the myocardium. Pretreatment withan undisclosed amount of uridine prevented the decrease in maximalpressure upon aortic ligation that is observed 2 hours after aorticstenosis. Meerson, F. C., In: Tr. Vseross. S'ezda Ter., Myasnikov, A. L.(ed.), Meditsina (publisher), Moscow, p. 27-32 (1966).

[0016] In another study, the use of glucose and uridine to controlcontractability and extensibility disturbances in the non-ischematizedcompartments of the heart after myocardial infarction were studied. Thedeficits in contractability and extensibility were reported to be due tosustained sympathetic nervous activity. The adddition of glucose oruridine in vitro restored contractability and extensibility of theisolated atrial tissue. Meerson, F. Z., et al., Kardiologiya 25:91-93(1985).

[0017] Despite the above results which were observed in isolated heartsor in situ organ preparations, the administration of uridine to intact(i.e., alive and free-running) animals has not been demonstrated to bebeneficial. Thus, while Eliseev, V. V., et al., Khim-Farm. Zh.19:694-696 (1985) (CA 103:82603k) disclose that uridine-5′-monophosphatehas a protective effect on rats with adrenaline-induced myocardialdystrophy, uridine was found to be relatively ineffective. Moreover,Williams, J. F., et al., Aust. N. Z. J. Med. 6:Supp. 2, 60-71 (1976),disclose that with rats developing hypertrophy of the heart, there wasno difference between rats which were treated with uridine compared withcontrols. Thus, except for rats which received continuous infusion ofuridine (Aussedat et al., supra), no beneficial effect on pathologyrelated to the heart has been demonstrated with uridine administration.

(2) Muscles

[0018] Exposure to uridine has also been found to enhance glucose uptakeand glycogen synthesis in isolated skeletal and cardiac muscle. Kypson,J., et al., Bioch. Pharmacol. 26:1585-1591 (1977). Uridine and inosinewere found to stimulate glucose uptake in isolated rat diaphragm muscle.However, only uridine increased glycogen synthesis. Both nucleosidesinhibited lipolysis in adipose tissue. Kypson, J., et al., J. Pharm.Exp. Ther. 199:565-574 (1976).

(3) Liver

[0019] Administration of cytidine and uridine has also been reported tobe effective in enhancing the regeneration of the liver in rats acutelypoisoned with carbon tetrachloride. Bushma, M. I., et al., Bull. Exp.Biol. Med. 88:1480-1483 (1980).

[0020] There have been a number of reports relating to the therapeuticadministration of nucleotides and RNA. The beneficial effects of RNA ornucleotides are probably due to their being broken down to individualribonucleosides by phosphatases. For example, injection of cytoplasmicRNA from the rat liver into mice during chronic poisoning with CCl₄reduced the mortality among the animals. Moreover, the number of foci ofnecrosis were reduced and the number of interlobular connective tissuefibers in the liver were increased. An increase in the mitotic activityof the liver cells was also observed. Chernukh, A. M., et al., Bull.Exp. Biol. Med. 70:1112-1114 (1970).

[0021] Administration of RNA, mixed nucleotides, or hydrocortisone,either alone or in various combinations, was found to increasetyrosine-alpha-ketoglutarate activity in rat liver. Administration ofRNA or nucleotides elevated enzymatic activity beyond the level attainedafter hydrocortisone administration alone. The authors speculated thatthe RNA or nucleotides may act via two mechanisms: first, a nonspecificstress effect, mediated through stimulation of adrenal steroid release,or secondly, through provision of limiting substrates for RNA synthesis.Diamondstone, T. I., et al., Biochim. Biophys. Acta 57:583-587 (1962).In a study on human patients with hepatic cirrhosis, administration ofcytidine and uridine improved insulin sensitivity in the cirrhoticpatient, but had no effect on insulin sensitivity in patients withoutliver disease. Ehrlich, H., et al., Metabolism 11:46-55 (1962).

[0022] In a study of repair after mechanical trauma in the liver, arapid, sustained increase in RNA content of cells at the border ofexperimentally induced trauma was observed. DNA concentrations in thetraumatized area began to rise on the third day after injury andcontinued to rise till the 10th day. The diabetic rat liver, incontrast, showed poor RNA and DNA contents. Increases in the tissuecontent of RNA and DNA around the traumatized site were delayed andstrongly depressed relative to nondiabetic livers. The failure of RNAsynthesis, which gives rise to poor wound healing in the diabetic liver,was attributed to deficient activity of the hexose monophosphate pathwayof glucose metabolism as observed in diabetics. Shah, R. V., et al., J.Anim. Morphol. Physiol. 25:193-200 (1978); Shah, R. V., et al., J. Anim.Morphol. Physiol. 21:132-139 (1974).

[0023] In another study, the availability of UDPG was found to berate-limiting for hepatic glycogen synthesis under some conditions. Whencultured hepatocytes were incubated with uridine, there was an increasein the incorporation of glucose into glycogen and tissue uridinenucleotide pools were expanded. When uridine was omitted from theincubation mixture, levels of UTP and UDPG dropped markedly during a 1hour incubation. Songu, E., et al., Metabolism 30:119-122 (1981). In astudy of patients with alcoholic hepatitis, a beneficial effect ofuridine-diphosphoglucose, when administered intramuscularly orintravenously, was found in biochemical indices as well as physiologicaland psychological symptoms. Thus, pyrimidine nucleosides are effectivein treatment of some forms of pathology of the liver.

(4) Diabetes

[0024] Nucleosides are also useful for the treatment of diabetes. Inexperimental diabetes, RNA synthesis is reduced in a number of tissues.Administration of oral sodium ribonucleate was found to increase therate of RNA biosynthesis in tissues of diabetic rats. Germanyuk, Y. L.,et al., Farmakol. Toksikol. 50-52 (1979). This effect is probably aresult of hydrolysis of the administered RNA to give individualribonucleotides and/or ribonucleosides. The failure of RNA synthesis inthe diabetic rat liver has been attributed to the deficient activity ofthe hexose monophosphate pathway of glucose metabolism in diabetes.Shah, R. V., et al., J. Anim. Morphol. Physiol. 25:193-200 (1978).

(5) Phospholipid Biosynthesis

[0025] Cytidine nucleotides have been implicated in phospholipidbiosynthesis. For example, Trovarelli, G., et al., NeurochemicalResearch 9:73-79 (1984), disclose that upon the intraventricularadministration of cytidine into the brain of rats, a measurable increasein the concentrations of all the nucleotides, CDP-choline,CDP-ethanolamine, and CMP occurred. The authors state that the lowconcentration of free cytidine nucleotides in nervous tissue likelylimits the rate of phospholipid biosynthesis.

(6) Brain

[0026] Administration of cytidine and uridine has also been reported tobe effective in the treatment of various neurological conditions inanimals. For example, Dwivedi et al., Toxicol. Appl. Pharmacol. 31:452(1978) disclose that uridine, administered by intraperitoneal injectionin mice, is an effective anticonvulsant, providing strong protectionGeiger et al., J. Neurochem 1:93 (1956) disclose that the functionalcondition of circulation-isolated cat brains perfused with washed bovineerythrocytes suspended in physiological saline remained normal for onlyabout 1 hour. If either the animal's liver was included in the perfusioncircuit, or cytidine and uridine were added to the perfusate, thefunctional condition of the brain remained good for at least 4 to 5hours. The cytidine and uridine tended to normalize cerebralcarbohydrate and phospholipid metabolism. The authors suggest that thebrain is dependent upon a steady supply of cytidine and uridine, whichare perhaps normally supplied by the liver.

[0027] Sepe, Minerva Medica 61:5934 (1970), disclose the effect of dailyintramuscular injections of cytidine and uridine in neurologicalpatients, most suffering from cerebrovascular disorders. Beneficialresults were obtained, particularly with respect to restoration of motorfunction, and in improving recovery after cranial trauma. No undesirableside effects were observed.

[0028] Jann et al., Minerva Medica 60:2092 (1969) disclose a study ofpatients with a variety of neurological disorders which were treateddaily with intramuscular injections of cytidine and uridine. Beneficialeffects were observed, particularly in cerebrovascular disordersinvolving motor function and mental efficiency. No undesirable sideeffects were observed.

[0029] Monticone et al., Minerva Medica 57:4348 (1966), disclose a studyof patients with a variety of encephalopathies which were treated withdaily intramuscular injections of cytidine and uridine. Beneficialeffects were found in most patients, particularly those withcerebrovascular disorders or multiple sclerosis. No undesirable sideeffects were observed.

[0030] One method that has heretofore been used, in effect, to introducecytidine equivalents into patients is the administration ofcytidine-diphosphocholine (CDP-choline). Cytidine-diphosphocholine, anintermediate in the biosynthesis of phosphatidyl choline (lecithin) isused therapeutically in Europe and Japan (under such names as Somazina,Nicholin, and Citicholine) for treating a variety of disorders.Therapeutic efficacy has been documented in central nervous systempathologies including brain edema, cranial trauma, cerebral ischemia,chronic cerebrovascular diseases, and Parkinson's disease. The mechanismunderlying the pharmacological actions of this compound is believed toinvolve support of phospholipid synthesis, restoration of thebiochemical “energy charge” of the brain, or a possible effect onneurotransmitter (particularly dopamine) function.

[0031] Examination of the fate of CDP-choline following itsadministration to animals or humans indicates that this compound is veryrapidly degraded, yielding cytidine, choline, and phosphate. After oraladministration, no intact CDP-choline enters the circulation, althoughplasma cytidine and choline concentrations rise. After intravenousinfection, breakdown to cytidine and choline occurs within about 30seconds. Therefore, it is difficult to attribute the therapeutic effectsof exogenous CDP-choline to the entry of this compound directly intocellular metabolism.

[0032] Therapeutic benefits in cerebral pathologies similar to thoseobtained with CDP-choline have been achieved following administration ofcytidine and uridine to humans and experimental animals. Therefore,CDP-choline appears to serve merely as an inefficient, expensive“prodrug” for cytidine, use of which perhaps hinders rather thanenhances the transport of cytidine to target tissues, compared toadministration of cytidine itself. Administration of choline by itselfdoes not result in the therapeutic benefits obtained afteradministration of either cytidine or CDP-choline. It would thus beadvantageous to develop methods for delivering cytidine to the brainthat are less expensive and/or more efficient than administration ofCDP-choline or cytidine itself.

[0033] Uridine-diphosphoglucose, uridine-diphosphoglucuronic acid, anduridine diphosphate also have been shown to improve certain aspects ofliver function. Since such phosphorylated compounds, as well asCDP-choline, must in general be dephosphorylated before they will entercells, administration of uridine, or derivatives of uridine, shouldrepresent a substantial improvement, in terms of both efficiency andcost, over the use of the phosphorylated pyrimidine derivatives.

(7) Immunulogical System

[0034] Cytidine and uridine may also have important influences on thefunction of the immune system. Kochergina et al. (Immunologiya0(5):34-37, 1986) disclose that administration of eithercytidine-5′-monophosphate or uridine-5′-monophosphate to micesimultaneously with an antigen (sheep red blood cells) results in astrong enhancement (relative to the response in animals treated withonly the antigen) of the humoral immune response to a subsequentchallenge with the antigen. Enhanced responsiveness of T-helperlymphocytes was reported to underlie this phenomenon. Thus, cytidine oruridine may be useful as adjuncts to improve the efficacy of vaccines,to improve the responsiveness of the immunce system in animmunocompromised patient, or to modify immune response in experimentalanimals. Van Buren et al., (Transplantation 40:694-697 (1985)) disclosethat dietary nucleotides are necessary for normal T-lymphocyte function;they did not, however, evaluate the influence of supra-normal amounts ofdietary or parenterally administered nucleotides or nucleosides.

[0035] In vivo, exogenous uridine itself is catabolized to a largeextent, rather than taken up and utilized for nucleotide synthesis.Gasser, T., et al., Science 213:777-778 (1981), disclose that theisolated, perfused rat liver degrades more than 90% of infused uridinein a single passage. Much of the uridine released by the liver in theportal vein is from degradation of liver nucleotides synthesized de novorather than from arterial uridine. This accounts for the poorutilization of administered uridine in peripheral tissues.

[0036] For example, Klubes, P., et al., Cancer Chemother. Pharmacol.17:236-240 (1986), disclose that after oral administration of 350 (mg/kgof uridine in mice, plasma levels of uridine were not perturbed. Incontrast, plasma levels of uracil, a catabolite of uridine, peaked at 50micro then declined and returned to normal after 4 h. Elevation ofplasma uridine levels was observed only after oral administration ofhigh doses of uridine (3500 mg/kg). However, such doses would be muchtoo high for an adult human since they would amount to about 200 g/dose.

[0037] A novel strategy for improving the bioavailability of cytidine oruridine after oral or parenteral administration is to administersderivatives of cytidine or uridine containing particular substituentswhich improve the pharmacokinetic or other pharmaceutical properties(e.g., transport across biological membranes) of these nucleosides.Properly chosen substituents, of which acyl substituents are best) willundergo enzymatic or chemical conversion back into cytidine or uridinefollowing administration.

[0038] Certain acylated uridine and cytidine derivatives are known, perse. Honjo, et al., in British Patent No. 1,297,398, describe N⁴, O^(2′),O^(3′), O^(5′)-tetraacylcytidines and a process for their preparation.The acyl substituents are those derived from fatty acids having fromthree to eighteen carbon atoms.

[0039] Beranek, et al., Collection Czechslovak Chem. Commun. (vol. 42,1977), p. 366-369, describe the preparation of 2′, 3′,5′-tri-O-acetylcytidine hydrochloride from cytidine by reaction withacetyl chloride in acetic acid.

[0040] Sasaki, et al., Chem. Pharm. Bull. (vol. 15, 1967), describe theacetylation of cytidine with acetic anhydride to form N4-acetylcytidine, 5′-O-acetylcytidine and N⁴, 5′-O-diacetylcytidine,among other compounds.

[0041] U.S. Pat. No. 4,022,963 to Deutsch, describes methods foracetylating all of the hydroxyl groups in the sugar portion of somenucleosides which include uridine, by a process including the additionof excess acetic anhydride.

[0042] Samoileva, et al., Bull. Acad. Sci. USSR Div. Chem. Sci. Vol. 30,1981, p. 1306-1310, disclose a method for synthesizing aminoacyl orpeptidyl derivatives of cytidine or cytidine monophosphate usinginsoluble polymeric N-hydroxysuccinimide. N⁴-BOC-alanyl cytidine wasprepared. The aminoacyl derivatives of cytidine were synthesized asprobes for studying the function of nucleases.

[0043] Japanese Patent Publications Nos. 51019779 and 81035196 assignedto Asahi Chemical Ind. KK describe methods for preparingN⁴-acyl-cytidines by reacting cytidine with acid anhydrides derived fromfatty acids containing 5 to 46 carbon atoms. The products are said to belipophilic ultra-violet absorbing agents and are also useful as startingcompounds in the preparation of anti-tumor agents.

[0044] Watanabe, et al., Angew. Chem., Vol. 78, 1986, p. 589 describemethods for selective acylation of the N⁴-amino group of cytidinewherein methanol is used as a solvent and acid anhydride as acylatingagent. Compounds prepared were N⁴-acetyl-, N⁴-benzoyl-, andN⁴-butyryl-cytidine.

[0045] Rees, et al., Tetrahedron Letters, Vol. 29, 1965, p. 2459-2465disclose methods for selective acylation of the 2′ position on theribose moiety of ribonucleosides. Uridine derivatives were preparedincluding 2′-O-acetyluridine, 2′-O-benzyluridine, and2′,5′-di-O-acetyluridine and other derivatives. The compounds wereprepared as intermediates in oligo-ribonucleotide synthesis.

OBJECTS OF THE INVENTION

[0046] While certain acylated derivatives of uridine and cytidine areknown and while the studies summarized above demonstrate that thepresence of uridine and cytidine is important to the amelioration of avariety of physiological and pathological conditions and that methodsfor enhancing the delivery of uridine and cytidine to animal tissue mayprovide an important source of those nucleosides, the art has heretoforefailed to provide methods for introducing uridine and cytidine intoanimal tissue at rates sufficiently high to reliably produce therapeuticeffects.

[0047] It is thus a primary object of this invention to identifypharmaceutically acceptable compounds which can be used efficiently todeliver pharmaceutically effective amounts of uridine and/or cytidine ortheir respective derivatives to animal tissue.

[0048] It is still a further object of this invention to provide afamily of uridine and cytidine derivatives which can be effectivelyadministered orally or parenterally and which have no untowardpharmaceutical effects.

[0049] It is still a further and related object of this invention toprovide a family of uridine and cytidine derivatives which, whenadministered to an animal, preferably humans, substantially improves thebioavailability of cytidine and uridine by enhancing the transport ofthose nucleosides across the gastrointestinal tract, the blood-brainbarrier, and other biological membranes and which allow sustaineddelivery of high levels of these ribonucleosides to animal tissues.

[0050] It is still a further and more specific object of this inventionto provide a family of cytidine and uridine derivatives for thetreatment of a variety of disorders including heart, muscle, plasma,liver, bone, diabetic, and neurological conditions.

[0051] These and other objects of the invention are achieved through theadministration of novel acyl derivatives of uridine and cytidine.

[0052] Broadly, the acyl derivatives of uridine comprise compoundshaving the formula (I)

[0053] wherein R₁, R₂, R₃, and R₄ are the same or different and each ishydrogen or an acyl radical of a metabolite, with the proviso that atleast one of said R substituents is not hydrogen, or a pharmaceuticallyacceptable salt thereof.

[0054] In one embodiment, acyl derivatives of uridine are those havingthe formula (II)

[0055] wherein R₁, R₂ and R are the same or different and each ishydrogen or an acyl radical of

[0056] (a) an unbranched fatty acid with 5 to 22 carbon atoms,

[0057] (b) an amino acid selected from the group consisting of glycine,L-forms of alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cystine, cysteine, aspartic acid,glutamic acid, arginine, lysine, histidine, carnitine, and ornithine,

[0058] (c) a dicarboxylic acid of 3 to 22 carbon atoms, or

[0059] (d) a carboxylic acid selected from one or more of the groupconsisting of glycolic acid, pyruvic acid, lactic acid, enolpyruvicacid, lipoic acid, pantothenic acid, acetoacetic acid, p-aminobenzoicacid, betahydroxybutyric acid, orotic acid, and creatine,

[0060] provided that at least one of said substituents R₁, R₂, and R₃ isnot hydrogen, and further provided that if any of said substituents R₁,R₂, and R₃ is hydrogen and if said remaining substituents are acylradicals of a straight chain fatty acid, then said straight chain fattyacid has 8 to 22 carbon atoms, or a pharmaceutically acceptable saltthereof. In particular, preferred dicarboxylic acids include succinic,fumaric, and adipic acids. In another embodiment, the objects of theinvention are also achieved with acyl derivatives of uridine comprisingcompounds having the formula (I) above wherein R₄ is not hydrogen.

[0061] The objects of the invention are also achieved by administrationof acyl derivatives of cytidine comprising compounds having the formula(III)

[0062] wherein R₁, R₂, R₃ and R₄ are the same or different and each ishydrogen or an acyl radical of a metabolite, with the proviso that atleast one of said R substituents is not hydrogen, or a pharmaceuticallyacceptable salt thereof.

[0063] Preferably, the cytidine derivatives are those having formula(III) wherein the R substituents are the same or different and each ishydrogen or an acyl radical derived from a carboxylic acid selected fromone or more of the group consisting of glycolic acid, pyruvic acid,lactic acid, enolpyruvic acid, an amino acid, a fatty acid of 2 to 22carbon atoms, a dicarboxylic acid, lipoic acid, pantothenic acid,acetoacetic acid, p-aminobenzoic acid, betahydroxybutyric acid, oroticacid, and creatine, or a pharmaceutically acceptable salt thereof.Preferred dicarboxylic acids include succinic, fumaric, and adipicacids.

[0064] The invention also includes pharmaceutical compositions whichcomprise one or more of the novel acylated ribonucleosides describedabove together with a pharmaceutically acceptable carrier. Thesecompositions may take the form of tablets, dragees, injectable solutionsand other forms.

[0065] Included among the novel pharmaceutical compositions of theinvention are those comprising certain known acyl derivatives of uridinetogether with a pharmaceutically acceptable carrier. Such compositionsinclude an acyl derivative of uridine having the formula (I) or (II)with substituents R₁, R₂, R₃ and R₄ as defined above, or apharmaceutically acceptable salt thereof. Preferred acyl derivatives ofuridine include 2′, 3′, 5′-tri-O-acetyl uridine, 2′, 3′,5′-tri-O-propionyl uridine, or 2′, 3′, 5′-tri-O-butyryl uridine.

[0066] The invention also includes pharmaceutical compositions ofcertain acyl derivatives of cytidine together with pharmaceuticallyacceptable carriers. Such acyl derivatives include those having theformula (III) with substituents R₁, R₂, R₃, and R₄ as described above,or a pharmaceutically acceptable salt thereof. Preferred acylderivatives of cytidine include 2′, 3′, 5′-tri-O-acetyl cytidine, 2′,3′, 5′-tri-O-propionyl cytidine, or 2′, 3′, 5′-tri-C-butyryl cytidine.

[0067] It has been found that the delivery of exogenous uridine orcytidine to animal tissue can be advantageously accomplished byadministering to the animal an effective amount of one or more of theacyl derivatives described above. It has further been found thatphysiological or pathological conditions of animal tissue may beadvantageously treated by supporting metabolic functions thereof byincreasing the bioavailability of uridine or cytidine to that tissue byadministering to an animal an effective amount of an acyl derivative asdescribed above.

[0068] The invention contemplates the use of these acyl derivatives fortreating a variety of physiological and pathological conditions,including treatment of cardiac insufficiency and myocardial infarction,treatment of liver disease or damage, muscle performance, treatment oflung disorders, diabetes, central nervous system disorders such ascerebrovascular disorders, Parkinson's disease, and senile dementias.The compounds of the invention improve the bioavailability of cytidineand uridine by enhancing the transport of these nucleosides across thegastrointestinal tract and other biological membranes, and prevent theirpremature degradation.

[0069] These advantageous uses of the acyl derivatives of the inventionare effected by administering compositions, as described above, of aneffective amount of one or more of these acyl derivatives and apharmaceutically acceptable carrier.

[0070] Administration of the acyl derivatives of cytidine and uridineoffer certain advantages over administration of the underivatizedcompounds. The acyl substituents can be selected to increase thelipophilicity of the nucleoside, thus improving its transport from thegastrointestinal tract into the bloodstream. The acylated derivativesare effective when administered orally. They are resistant to catabolismby nucleoside deaminases and nucleoside phosphorylases in the intestine,liver, other organs, and the bloodstream. Thus, administration of theacylated derivatives of the invention, either orally or parenterally,allows sustained delivery of high levels of these ribonucleosides to thetissues of an animal.

DESCRIPTION OF THE FIGURES

[0071]FIG. 1: this figure shows the basal heart work output forundamaged (received only saline) rats, rats with experimental myocardialdamage but untreated (received only saline) and rats treated withtriacetyluridine (TAU) and triacetylcytidine (TAC) after experimentalmyocardial damage.

[0072]FIG. 2: this figure shows the basal left ventricular systolicpressure of control rats, untreated rats, and rats treated with TAU andTAC after experimental myocardial damage.

[0073]FIG. 3: this figure shows the basal maximum rate of ventricularcontraction of control rats, untreated rats, and rats treated with TAUand TAC after experimental myocardial damage.

[0074]FIG. 4: this figure shows the basal maximum rate of ventricularrelaxation of control rats, untreated rats, and rats treated with TAUand TAC after experimental myocardial damage.

[0075]FIG. 5: this figure shows the basal heart rate of control rats,untreated rats, and rats treated with TAU and TAC after experimentalmyocardial damage.

[0076]FIG. 6: this figure shows the maximum heart work output of controlrats, untreated rats, and rats treated with TAU and TAC andnorepinephrine after experimental myocardial damage.

[0077]FIG. 7: this figure shows the maximum left ventricular systolicpressure of control rats, untreated rats, and rats treated with TAU andTAC and norepinephrine after experimental myocardial damage.

[0078]FIG. 8: this figure shows the maximum rate of ventricularcontraction (maximum) of control rats, untreated rats, and rats treatedwith TAU and TAC and norepinephrine after experimental myocardialdamage.

[0079]FIG. 9: this figure shows the maximum rate of ventricularrelaxation (maximum) of control rats, untreated rats, and rats treatedwith TAU and TAC and norepinephrine after experimental myocardialdamage.

[0080]FIG. 10: this figure shows the heart rate (maximum) of controlrats, untreated rats, and rats treated with TAU and TAC andnorepinephine after experimental myocardial damage.

[0081]FIG. 11: this figure shows plasma BSP clearance in rats with liverdamage treated with TAC and TAU or with water (control).

DESCRIPTION OF THE PREFERRED EMBODIMENTS Definition of Terms

[0082] A “metabolite” is a chemical compound that is formed by, orparticipates in, a metabolic reaction. In the context of thisapplication, metabolites refer, in particular, to carboxylic acids knownto be synthesized within the human body, and also naturally occurring(but perhaps synthesized rather than extracted) substituents that mightbe derived from other animal or plant sources. The limiting criteria arethat the compound should be substantially nontoxic and biocompatible,and should readily enter into metabolic pathways in vivo, so as topresent essentially no toxicity during long-term consumption in thedoses proposed. It is preferable that the substituents be metabolizedrather than excreted intact (or conjugated through detoxificationreactions), as concentration of carboxylic acids within the kidney maylead to undesirable excessive acidity. Therefore, carboxylic acids thatnormally or easily participate in intermediary, catabolic, or anabolicmetabolism are preferred substituents. Preferably such carboxylic acidsare of molecular weight less than 1000 Daltons.

[0083] “Pharmaceutically acceptable salts” means salts withpharmaceutically acceptable acid addition salts of the nucleosidederivatives of the invention. Such acceptable acids include, but are notlimited to, sulfuric, hydrochloric, or phosphoric acids.

[0084] “Coadministered” means that each of at least two acyl nucleosidederivatives are administered during a time frame wherein the respectiveperiods of pharmacological activity overlap.

[0085] “Acyl derivatives” are derivatives of cytidine or uridine inwhich a substantially nontoxic organic acyl substituent derived from acarboxylic acid is attached to one or more of the free hydroxyl groupsof the ribose moiety of cytidine or uridine with an ester linkage,and/or where such a substituent is attached to a primary or secondaryamine in the pyrimidine ring of cytidine or uridine, with an amidelinkage. Such acyl substituents include, but are not limited to, thosederived from acetic acid, fatty acids, amino acids, lipoic acid,glycolic acid, lactic acid, enolpyruvic acid, pyruvic acid, orotic acid,acetoacetic acid, beta-hydroxybutyric acid, creatinic acid, succinicacid, fumaric acid, adipic acid, and p-aminobenzoic acid. Preferred acylsubstituents are compounds which are normally present in the body,either as dietary constituents or as intermediary metabolites, and whichare essentially nontoxic when cleaved from the ribonucleoside in vivo.

[0086] “Fatty acids” are aliphatic carboxylic acids having 2 to 22carbon atoms. Such fatty acids may be saturated, partially saturated orpolyunsaturated.

[0087] “Amino acids” include, but are not limited to, glycine, the Lforms of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine,proline, hydroxyproline, serine, threonine, cysteine, cystine,methionine, tryptophan, aspartic acid, glutamic acid, arginine, lysine,histidine, ornithine, and hydroxylysine. However, the invention is notso limited, it being within the contemplation of the invention toinclude other naturally occurring amino acids.

[0088] The lipophilic acyl derivatives of uridine and cytidine areuseful for enhancing the transport of the nucleotides across thegastrointestinal tract in animals. Foremost among such animals arehumans. However, the invention is not intended to be so limited, itbeing contemplated that all animals may be treated with the acylderivatives of the present invention with attendant beneficial effect.

[0089] Although the invention is not bound by a specific mechanism ofaction, the compounds of the present invention appear to effect theirbeneficial activity by increasing the bioavailability of cytidine anduridine, and thereby, improving tissue regeneration, repair,performance, resistance to damage, and adaptation to physiologicaldemand. They may work, as well, by increasing the bioavailability ofnucleoside anabolites, e.g., nucleotides or nucleotide-derivedcofactors. Administration of the nucleosides per se increases theirbioavailability but, due to rapid catabolism, this may not result insignificant elevation of nucleotide levels; i.e., one doesn'tnecessarily get an increase in plasma levels because at lower nucleosidelevels there is rapid uptake by the cells whereas at higher levels thereis saturation and the excess is degraded. The invention is believed towork by delivering a sustained supply of nucleoside at lower levels.

[0090] Preferred acyl derivatives of cytidine or uridine for enhancingtransport across biological membranes are those which are morelipophilic than are the parent nucleosides. In general, lipophilic acylnucleoside derivatives have acyl substituents which are nonpolar (asidefrom the carboxylate group). Such acyl substituents are derived fromacids including, but not limited to, acetic acid, lipoic acid, and fattyacids. One of ordinary skill in the art can determine whether aparticular acyl nucleoside derivative is more lipophilic than theunderivatized nucleoside using standard techniques, i.e., comparison ofthe partition coefficients determined in water-octanol mixtures.Following passage of the acylated nucleoside derivative from thegastrointestinal tract into the bloodstream or across other biologicalmembranes, the acyl substituents are cleaved by plasma and tissueesterases (or amidases) to give the free nucleosides.

[0091] The rate of removal of the acyl substituents in vivo is afunction of the specificity of plasma and tissue deacylating enzymes(primarily esterases or amidases). Acyl substituents attached to anamine group in the pyrimidine ring of cytidine or uridine with an amidelinkage are cleaved more slowly than are substituents attached tohydroxyl groups of ribose with an ester linkage.

[0092] It is also possible to prepare acyl nucleoside derivatives whichcontain polar and nonpolar acyl substituents. The polar acyl substituentwill retard passage of the nucleoside derivative from thegastrointestinal tract, allowing for a more sustained delivery of thecompound into the bloodstream after a single dose. The polar group maybe cleaved by esterases, amidases, or peptidases present in theintestinal tract to give a nucleoside with a nonpolar acyl substituentwhich may then efficiently enter the circulation. Polar acylsubstituents may be chosen by one of ordinary skill in the art, withoutundue experimentation, which are cleaved at a faster rate than arenonpolar acyl substituents.

[0093] The acyl derivatives are also less susceptible to degradation ofthe nucleoside moiety by enzymes in plasma and non-target tissues, andare also less susceptible to elimination from the bloodstream via thekidneys. For parenteral injection, acyl derivatives with polar acylsubstituents, which are therefore water soluble yet resistant topremature degradation or elimination, may be used with advantage.Preferred acyl derivatives in such application include glycolate andlactate and those derived from amino acids with polar side chains.

Therapeutic Uses

[0094] Administration of the acyl derivatives of cytidine may be usefulin treating lung disorders, including infant respiratory distresssyndrome (IRDS), and in metabolic disorders that affect pulmonaryfunction. The acyl derivatives appear to support or enhance phospholipidbiosynthesis and surfactant formation in the lung. The main component ofthe surfactant, phosphatidyl choline, is derived from cytidinediphosphocholine. Thus, administration of the acylated form of cytidinewill support or augment the capacity of pneumocytes to synthesizephospholipids and generate surfactant. The beneficial effects ofcytidine acyl derivatives may be enhanced by coadministering uridineacyl derivatives.

[0095] In addition, administration of the acyl derivative of cytidinemay be useful in treatment of neural disorders. The acyl derivatives mayexert their activity by restoring or maintaining brain phospholipidcomposition during or after a period of cerebral hypoxia or stroke.Administration of the acyl derivative of cytidine may also be useful inslowing the onset or progression of degenerative disorders. Disorderssuch as cerebrovascular disorders, Parkinson's disease, and cerebralataxia have been linked to phospholipid levels. The acyl derivatives ofuridine may be advantageously coadministered with the acyl derivative ofcytidine to enhance its effect.

[0096] Administration of the acyl derivatives of cytidine and uridinemay be effective for the treatment of cerebrovascular dementias andParkinson's disease. Cerebrovascular dementias and Parkinson's diseasecause gradual, generally symmetric, relentlessly progressive wastingaway of the neurons. Cerebral ataxia is characterized by loss of nervecells principally affecting the Purkinje cells.

[0097] Therefore, administration of the acyl derivatives of cytidine anduridine may exert their activity by enhancing phospholipid biosynthesisand thereby ameliorating the effects of cerebrovascular disorders,Parkinson's disease and cerebral ataxia.

[0098] The invention also relates to treatment of physiological orpathophysiological conditions where the body's capacity to synthesizenucleic acids is suboptimal. These conditions include diabetes,senescence, and adrenal insufficiency. Administration of the acylderivatives of cytidine and uridine may provide a beneficial effect byproviding a sustained delivery of high levels of cytidine and uridine togive sufficient pools of nucleotides which are necessary for thebiosynthesis of enzymes crucial for cellular self regeneration.

[0099] Although the invention is not bound by any one mode of action,the compositions of the present invention, in and of themselves, appearto act by enhancing nucleotide and nucleic acid synthesis and proteinsynthesis by providing nucleosides under conditions wherein de novosynthesis is not sufficient for supporting optimal rates of nucleotideand nucleic acid synthesis. Thus, the compounds may find utility intreatment of cardiac insufficiency, myocardial infarction, liver diseaseincluding cirrhosis, and by reversing the pathological effects ofdiabetes by accelerating nucleic acid synthesis and thereby proteinsynthesis.

[0100] The acyl derivatives of uridine and cytidine may be administeredto improve ventricular function after myocardial infarction or intreating or preventing cardiac insufficiency. The acyl nucleosidederivatives of the invention, which appear to support the cellularmechanisms involved in calcium sequestration, and which thereby preserveor support cellular ATP regeneration, may have significant therapeuticvalue in preventing or treating some of the deleterious effects ofmyocardial insults.

[0101] The compositions of the present invention may be coadministeredwith drugs which are used to treat cardiac insufficiency, e.g.,digitalis, diuretics and catecholamines.

[0102] It is possible to attenuate load-induced myocardial damage, andto promote stable hyperfunction by providing to the heart substancesthat support the biochemical processes involved in calcium sequestrationand RNA biosynthesis. Uridine and cytidine are useful compounds in thiscontext. Uridine has been reported to be relatively ineffective insupporting myocardial hyperfunction in vivo; however, this may bebecause uridine is rapidly degraded by plasma and tissue enzymes, whichconsequently prevents its utilization by the heart. The invention isbased partly on the finding that it is possible to improve delivery ofuridine to the heart by administering the acyl derivatives whichgradually release free uridine into the bloodstream over an extendedperiod of time.

[0103] The acyl derivatives of uridine may be administered to treathypoxia or anoxia. These acyl derivatives appear to act by enhancingbiosynthesis of uridine diphosphoglucose, a necessary intermediate inglycogen synthesis, to improve tissue resistance to hypoxia or anoxiaand preserve the functional capacity of tissues, in particular cardiac.Uridine acyl derivatives may be used for the treatment of hypoxia,anoxia, ischemia, excessive catecholaminergic stimulation, and digoxintoxicity.

[0104] The compounds of the present invention may also find utility incountering some of the long term complications of diabetes, whichinclude neuropathies, arteriopathies, increased susceptibility to bothcoronary arteriosclerosis and myocardial infarction, and blindness.Since de novo nucleotide synthesis is suppressed in diabetes, exogenousacyl derivatives of nucleosides have therapeutuc value in treatingdiabetes. In addition, the derivatized forms of the nucleosides may beadministered to provide sufficient pools of nucleosides necessary forthe biosynthesis of enzymes crucial for cellular self regeneration.Thus, the invention also relates to methods for treating, for example,diabetic liver, or vascular disease by administering the acyl nucleosidederivatives of the invention. The acyl nucleoside derivatives are alsouseful in supporting or enhancing muscular hypertrophy or hyperfunctionin response to increased demand. Such demand may occur after sustainedmuscular exertion.

[0105] Preferred acyl substituents include acetyl, propionyl, andbutyryl groups. Preferred acyl nucleoside derivatives include 2′, 3′,5′-tri-O-acetyl cytidine, 2′, 3′, 5′-tri-O-acetyl uridine, 2′, 3′,5′-tri-O-propionyl uridine, 2′, 3′, 5′-tri-O-propionyl cytidine, 2′, 3′,5′-tri-O-butyryl cytidine and 2′, 3′, 5′-tri-O-butyryl uridine. It canbe advantageous to coadminister acyl derivatives of both cytidine anduridine.

[0106] Typical dosage forms are equivalent to 10 to 3000 mg of cytidineand/or uridine in the form of their acyl derivatives or thepharmaceutically acceptable salt thereof, 1 to 3 times per day. Thiscorresponds to, for example, 15 to 4500 mg of 2′, 3′, 5′-tri-O-acetylcytidine and 2′, 3′, 5′-tri-O-acetyl uridine.

[0107] For treatment of cardiac insufficiency, myocardial infarction andthe consequences of hypertension, a composition comprising 25 to 100mole percent of the acyl derivative of uridine may be coadministeredtogether with 75 to 0 mole percent of the acyl derivative of cytidinewith the proviso that the amounts of the acyl derivatives or cytidineand uridine do not exceed 100 mole percent. For example, 1125-4500 mg of2′, 3′, 5′-tri-O-acetyluridine may be administered with 0-3475 mg of 2′,3′, 5′-tri-O-acetylcytidine.

[0108] For treatment of cerebrovascular disorders, diabetes, liverdamage and disease, and to increase muscle performance, a compositioncomprising 25 to 75 mole percent of the acyl derivative of uridine maybe coadministered together with 75 to 25 mole percent of the acylderivative of cytidine with the proviso that the amount of the acylderivatives of uridine and cytidine do not exceed 100 mole percent. Forexample, 1125-3375 mg of 2′, 3′, 5′-tri-O-acetyluridine may becoadministered with 1125-3375 mg of 2′, 3′, 5′-tri-O-acetyl cytidine.

[0109] For treatment of respiratory distress syndrome, 25 to 100 molepercent of the acyl derivative of cytidine may be coadministered with 75to 0 mole percent of the acyl derivative of uridine with the provisothat the amounts of the acyl derivatives of uridine and cytidine do notexceed 100 mole percent. For example, 1125-4500 mg of 2′, 3′,5′-tri-O-acetyl cytidine may be coadministered together with 0-3375 mgof 2′, 3′, 5′-tri-O-acetyl uridine.

Examples of Therapeutic Administration Cardiac Insufficiency

[0110] Acyl derivatives of cytidine and uridine are useful in thetreatment of several varieties of cardiac insufficiency. They areeffective in supporting sustained compensatory hyperfunction in the caseof increased load upon the heart in hypertension, for example, orespecially in supporting the function of the surviving portions of theheart after a myocardial infarction. In this latter situation, a mixtureof acyl derivatives of cytidine and uridine may be given as soon afterthe onset of the infarction as possible, followed by chronic oraladministration of a suitable formulation of acyl derivatives of thesenucleosides in doses of approximately 0.5 to 3.0 grams per day of each.These compounds may be used advantageously in conjunction withconventional treatments of myocardial infarction. The nucleosidederivatives have the unique advantage of protecting the heart againstdamage secondary to overload, hypoxia, or catecholamines withoutreducing the functional capacity of the heart, since they act byenhancing the metabolic integrity of the myocardium, in particular byimproving calcium handling. The nucleoside derivatives may also beadministered prophylactically to patients at risk for myocardialinfarction or cardiac insufficiency.

[0111] For treatment of chronic cardiac insufficiency, which leads tocongestive heart failure, acyl derivatives of cytidine and uridine maybe administered orally in doses ranging from 0.5 to 3 grams per day ofeach nucleoside. The nucleosides may be used in conjunction with otheragents such as digitalis derivatives or diuretics. In addition toimproving myocardial function directly, the nucleoside derivativesreduce digitalis toxicity without impairing its clinical efficacy.

Diabetes

[0112] In many tissues of diabetic subjects, cellular pyrimidinenucleotide levels are reduced; this may contribute to some of thelong-term complications of diabetes, including arteriopathies,neuropathies, and decreased resistance of the myocardium to mechanicalor biochemical stress. These complications are related to malfunctionsin tissue calcium handling, in which pyrimidine nucleotides play keyroles. It has been reported that daily intramuscular injection ofcytidine and uridine reverses the depression in peripheral nerveconduction velocity in diabetic humans (C. Serra, Rif. Med. 85:1544[1971]). It is preferable to administer acyl derivatives of cytidine anduridine orally in suitable formulations. Doses equivalent to 0.5 to 3grams of cytidine and uridine are administered daily, in conjunctionwith conventional anti-diabetic treatments. The nucleotide derivativesare particularly useful in non-insulin dependent diabetes.

Neurological Disorders

[0113] In the treatment of the consequences of cerebrovasculardisorders, e.g., stroke and chronic or acute cerebrovascularinsufficiency, acyl derivatives of cytidine and uridine, particularlythose formulated to pass through the blood-brain barrier after oraladministration, may be administered in oral doses ranging from 0.5 to3.0 grams of each nucleoside per day for at least several months.

[0114] In Parkinson's disease, acyl cytidine derivatives areparticularly useful, and may be given in conjunction with theconventional treatment of choice, L-DOPA. The cytidine derivatives,administered in oral doses of 0.5 to 3.0 grams per day, may permitsatisfactory clinical maintenance on reduced dosages of L-DOPA, which isadvantageous because L-DOPA has undesirable side effects.

Methods of Preparation of the Compounds

[0115] The acyl derivatives of the invention may be prepared by thefollowing general methods. When the acyl substituent has groups whichinterfere with the acylation reactions, e.g., hydroxyl or amino groups,these groups may be blocked with protecting groups, e.g.,t-butyldimethylsilyl esters or t-BOC groups, respectively, beforepreparation of the anhydride. For example, lactic acid may be convertedto 2-(t-butyldimethylsiloxy)propionic acid witht-butyldimethylchlorosilane, followed by hydrolysis of the resultingsilyl ester with aqueous base. The anhydride may be formed by reactingthe protected acid with DCC.

[0116] In the case of amino acids, the N-t-BOC derivative may beprepared, using standard techniques, which is then converted to theanhydride with DCC.

[0117] Derivatives containing acyl substituents with more than onecarboxylate group (e.g., succinate, fumarate, or adipate) are preparedby reacting the acid anhydride of the desired dicarboxylic acid with a2′-deoxyribonucleoside in pyridine.

[0118] For example, the 2′, 3′, 5′-tri-O-acyl derivatives of uridine maybe prepared by a modified procedure disclosed by Nishizawa et al.,Biochem. Pharmacol. 14:1605 (1965). To one equivalent of uridine inpyridine is added 3.1 equivalents of an acid anhydride (aceticanhydride, butyric anhydride, etc.), and the mixture heated to 80-85° C.The triacyl derivative may then be isolated using standard techniques.Alternatively, uridine may be treated with 3.1 equivalents of a desiredacid chloride (acetyl chloride, palmitoyl chloride, etc.) in pyridine atroom temperature (See Example V).

[0119] The 5′ acyl derivative of uridine may be prepared according toNishizawa et al. by reacting uridine with 1 equivalent of the acidanhydride of the desired acyl compound in pyridine at room temperature.The reaction is then heated to 80-85° C. for two hours, cooled, and the5′-acyl derivative isolated by standard techniques and purified bychromatography. Alternatively, the 5′-acyl derivative of uridine may beprepared by treating uridine, in pyridine and DMF at 0° C., with 1equivalent of the acid chloride derived from the desired acyl compound.The 5′-acyl derivative of uridine may then be isolated by standardtechniques and purified by chromatography (see Example VI).

[0120] The 2′, 3′-diacyl derivatives of uridine may be prepared by aprocedure adapted from Baker et al., J. Med. Chem. 22:273 (1979). The5′-hydroxyl group is selectively protected with 1.2 equivalents oft-butyldimethylsilyl chloride in DMF containing imidazole, at roomtemperature. The 5′-t-butyldimethylsilyl derivative of uridine isisolated by standard techniques, then treated with 2.1 equivalents ofthe acid anhydride of the desired acyl compound in pyridine at 0-5° C.The resulting 5′-t-butyl dimethylsiloxy-2′, 3′-diacyl uridine is thentreated with tetrabutylammonium fluoride and the 2′, 3′-diacylderivative of uridine is isolated by standard techniques (see ExampleVII).

[0121] The secondary amine of 2′, 3′, 5′-tri-O-acyl uridine may then beacylated according to Fujii et al., U.S. Pat. No. 4,425,335, whichinvolves treatment with 1.1 equivalents of an acid chloride in anaprotic solvent containing 1-5 equivalents of an organic base, e.g.,aromatic amines such as pyridine, trialkylamines, orN,N-dialkylanilines. Using this procedure, a tetraacyl derivative ofuridine may be prepared which has an acyl substituent on the amino groupwhich is different from the acyl substituents at the 2′, 3′ and 5′hydroxy groups (see Example VIII).

[0122] The 2′, 3′, 5′-tri-O-acyl derivatives of cytidine may be preparedaccording to a method adapted from Gish et al., J. Med. Chem. 14:1159(1971). For example, cytidine hydrochloride may be treated with 3.1equivalents of the desired acid chloride in DMF. The 2′, 3′,5′-tri-O-acyl derivative may then be isolated using standard techniques(see Example IX).

[0123] The 5′-acyl derivative of cytidine may be prepared according toGish et al., supra, by treatment of cytidine hydrochloride with 1.1equivalents of an acid chloride in DMF, followed by isolation of 5′-acylcytidine by standard techniques (see Example X).

[0124] Selective acylation of the N⁴-amine of cytidine accomplishedaccording to the procedure disclosed by Sasaki et al., Chem. Pharm.Bull. 15:894 (1967). This involves treatment of cytidine with 1.5equivalents of an acid anhydride in pyridine and DMF. The N⁴-acylderivative of cytidine may then be isolated by standard techniques (seeExample XI).

[0125] Alternatively, the N⁴-acyl derivative of cytidine may be preparedby treatment of cytidine with an acyl anhydride in pyridine or a mixtureof pyridine and DMF. Another procedure for the selective preparation ofN⁴-acyl cytidine involves selective acylation with an acid anhydride ina water-water miscible solvent system according to Akiyama et al., Chem.Pharm. Bull. 26:981 (1978) (see Example XI).

[0126] Tetraacyl cytidine derivatives, where all the acyl groups are thesame, may be prepared by treating cytidine with at least 4 molarequivalents of an acid anhydride in pyridine at room temperature. Thetetraacyl cytidine may then be isolated using standard techniques (seeExample XII).

[0127] To prepare compounds in which the acyl substituent on the N⁴amino group is different from the acyl substituents on the hydroxylgroups of the ribose ring (e.g., N⁴-palmitoyl 2′, 3′, 5′-tri-O-acetylcytidine), the desired acyl substituent is selectively attached to theN⁴ amino group as described above, and then the hydroxyl groups areacylated with their intended substituents. Alternatively, thesubstituents on the ribose moiety may be attached prior to attachment ofthe substituent of the N⁴ amino group, again using methods describedabove.

[0128] Compositions within the scope of this invention include allcompositions wherein each of the components thereof is contained in anamount effective to achieve its intended purpose. Thus, the compositionsof the invention may contain one or more acyl nucleoside derivatives ofuridine or cytidine in amounts sufficient to result, uponadministration, in increased plasma or tissue levels of cytidine oruridine and the acyl derivatives thereof, which thereby produce theirdesired effect.

[0129] In addition to the pharmacologically active compounds, the newpharmaceutical preparations may contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into the preparationswhich may be used pharmaceutically. Preferably, the preparations,particularly those preparations which can be administered orally andwhich can be used for the preferred type of administration, such astablets, dragees, and capsules and also preparations which can beadministered rectally, such as suppositories, as well as suitablesolutions for administration by injection or orally, contain about from0.1 to 99%, preferably from about 10-90%, of the active compound(s),together with the excipient.

[0130] The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral usecan be obtained by combining the active compound(s) with solidexcipients, optionally grinding a resulting mixture and processing themixture of granules, after adding suitable auxiliaries, if desired ornecessary, to obtain tablets or dragee cores.

[0131] Suitable excipients are, in particular, fillers such as sugars,for example lactose or sucrose, mannitol or sorbitol, cellulosepreparations and/or calcium phosphates, for example tricalcium phosphateor calcium hydrogen phosphate, as well as binders such as starch paste,using, for example, maize starch, wheat starch, rice starch, potatostarch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethyl cellulose, and/or polyvinyl pyrrolidone.It desired, disintegrating agents may be added such as theabove-mentioned starches and also carboxymethylstarch, cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such assodium alginate. Auxiliaries are, above all, flow-regulating agents andlubricants, for example, silica, talc, stearic acid or salts thereof,such as magnesium stearate or calcium sterate, and/or polyethyleneglycol. Dragee cores are provided with suitable coatings which, ifdesired, are resistant to gastric juices. For this purpose, concentratedsugar solutions may be used, which may optionally contain gum arabic,talc, polyvinyl pyrrolidone, polyethylene glycol and/or titaniumdioxide, lacquer solutions and suitable organic solvents or solventmixtures. In order to produce coatings resistant to gastric juices,solutions of suitable cellulose preparations such as acetylcellulosephthalate or hydroxylpropylmethylcellulose phthalate are used. Dyestuffs or pigments may be added to the tablets or dragee coatings, forexample, for identification or in order to characterize differentcombinations of compound doses. Other pharmaceutical preparations whichcan be used orally include push-fit capsules made of gelatin, as well assoft-sealed capsules made of gelatin and a plasticizer such as glycerolor sorbitol. The push-fit capsules contain the active compound(s) in theform of granules which may be mixed with fillers such as lactose,binders such as starches and/or lubricants such as talc or magnesiumsterate, and, optionally, stabilizers. In soft capsules, the activecompounds are preferably dissolved or suspended in suitable liquids suchas fatty oils, liquid paraffin, or polyethylene glycols. In addition,stabilizers may be added.

[0132] Possible pharmaceutical preparations which can be used rectallyinclude, for example suppositories which consist of a combination ofactive compounds with a suppository base. Suitable suppository basesare, for example, natural or synthetic triglycerides, paraffinhydrocarbons, polyethylene glycols or higher alkanols. In addition, itis also possible to use gelatin rectal capsules which consist of acombination of the active compounds with a base. Possible base materialsinclude, for example, liquid triglycerides, polyethylene glycols, orparaffin hydrocarbons.

[0133] Suitable formulations for parenteral administration includeaqueous solutions of the active compounds in water soluble form, forexample, water soluble salts. In addition, suspensions of the activecompounds as appropriate oily injection suspensions may be administered.Suitable lipophilic solvents or vehicles include fatty oils, forexample, sesame oil, or synthetic fatty acid esters, for example, ethyloleate or triglycerides. Aqueous injection suspensions may includesubstances which increase the viscosity of the suspension which include,for example, sodium carboxymethylcellulose, sorbitol, and/or dextran.Optionally, the suspension may also contain stablizers.

[0134] The following examples are illustrative, but not limiting, of themethods and compositions of the present invention. Other suitablemodifications and adaptations of the variety of conditions andparameters normally encountered in clinical therapy and which areobvious to those skilled in the art and are within the spirit and scopeof this invention.

EXAMPLES Example I Comparison of the Bioavailability of Uridine and AcylUridine Derivatives in Rats

[0135] Silastic catheters were implanted into the right jugular veins ofanesthetized male F344 rats (Retired Breeders, 450-500 grams). Threedays later, blood samples were withdrawn without disturbing the rats.Basal blood samples were taken and the animals were divided into fourgroups, each containing four rats. Each group received a different oneof each of the following compounds; uridine, 2′, 3′, 5′-tri-O-acetyluridine, cytidine, or 2′, 3′, 5′-tri-O-acetyl cytidine. The compoundswere given in equimolar dosages (0.28 moles/kg) by intubation into thestomach. At intervals of 0.5, 1, 2, 3, and 4 hours after administration,blood samples (0.3 ml) were withdrawn and processed for subsequent assayof cytidine or uridine content by HPLC. In the rat, plasma levels ofuridine were significantly higher (5 to 10 fold) for at least four hoursfollowing ingestion of tri-O-acetyl uridine than after ingestion of anequimolar dose of uridine.

Example II

[0136] Comparison of the Bioavailability of Uridine and Acyl UridineDerivatives in Humans

[0137] After an overnight fast, a basal venous blood sample waswithdrawn from a human subject and then 0.76 moles/kg (28 mg/kg—2 gramsin a 70 kg subject) of tri-O-acetyl uridine was ingested along with 100ml of water. Blood samples (0.5 ml) were withdrawn at intervals of 1, 2,3, and 4 hours after ingestion of the compound and were processed forsubsequent determination of plasma uridine content by HPLC. On aseparate day, the same procedure was carried out, except that an equalmolar dose (18 mg/kg—1.3 grams in a 70 kilogram subject) of uridine wasingested instead of the acyl derivative. The plasma level of uridine wassubstantially higher following ingestion of tri-O-acetyl uridine thanafter ingestion of the equal molar dose of uridine. Uridine levels weresustained in the useful therapeutic range (greater than 10 micromolar)for at least four hours after oral administration of tri-O-acetyluridine. After administration of oral uridine, plasma levels of thenucleoside exceeded 10 micromolar at only one point in time (two hours).

Example III

[0138] Restoration of Depressed Myocardial Function with AcylatedPyrimidine Ribonucleosides

[0139] The experiment described within this example was designed todetermine whether providing exogenous triacetyl uridine and triacetylcytidine could help to restore pump function in the ventricularmyocardium after experimental depression of ventricular function.

[0140] Experimental myocardial damage was induced in anesthetized(Nembutal, 50 mg/kg i.p.) male F344,rats (250 grams) by constricting theabdominal aorta to an internal diameter of 0.67 mm, followed byinjection of a single dose of isoproterenol hydrochloride (5 mg/kgs.c.). A mixture of triacetyl cytidine and triacetyl uridine (590 mg/kgof each) was administered immediately after, and again 1 hour and 20hours after aorta constriction and administration of isoproterenol. Someanimals received injections of saline instead of the acetylatednucleotides (untreated), and a group of animals also received salineinjections but were not subjected to aorta constriction or treatmentwith isoproterenol (controls). Ventricular function was determined 24hours after aortic constriction. Animals were anesthetized with sodiumpentobarbital (50 mg/kg i.p.), and a catheter was implanted in the rightjugular vein for administration of norepinephrine. A second catheter(Intramedic PE-50) was inserted into the left ventricle of the heart viathe right carotid artery. Left ventricular systolic pressure (LVSP), themaximum rate of ventricular contraction and relaxation (+dP/dT and-dP/dT, respectively) and heart rate (He) were measured directly viathis catheter, using a Statham-type pressure transducer interfaced to aStoelting Physioscribe II polygraph. Values of these parameters wererecorded, before and after i.v. administration of 0.1 ml ofnorepinephrine bitartrate at concentrations of 10⁻⁶, 10⁻⁵, and 10⁻⁴M.Electrocardiograms were also recorded with this apparatus, usingstainless steel needle electrodes inserted subcutaneously in theforelimbs. Heart work output was calculated as the product ofventricular systolic pressure and heart rate.

[0141] Aorta constriction in conjunction with isoproterenoladministration resulted in substantial decrements in myocardialperformance compared to intact controls. Left ventricular systolicpressure, +dP/dT, −dP/dT, and heart work output were all significantlydepressed (Table 1; FIGS. 1-4). In the animals that received acetylatedpyrimidine nucleosides after aorta constriction and administration ofisoproterenol, all of these parameters were significantly restoredtoward normal, compared to animals treated only with isoproterenol(FIGS. 1-4). Heart rate was also depressed after experimental myocardialdamage (FIG. 5). TABLE 1 Basal Heart Performance LVSP HR +dP/dT −dP/dTHR × LVSP TREATMENT (mmHg) (bpm) (mmHg/sec) (mmHg/sec) (mmHg/min)Control 141 ± 11 386 ± 46 6000 ± 348 5640 ± 528 55,766 ± 10,407 AC +Saline  107 ± 14* 283 ± 44  4080 ± 600*  3120 ± 840* 32,633 ± 9,115*AC + TAU & TAC 158 ± 9  398 ± 28 6000 ± 480 5640 ± 300 63,518 ± 6,624

[0142] TABLE 2 Maximal Heart Performance LVSP HR +dP/dT −dP/dT HR × LVSPTREATMENT (mmHg) (bpm) (mmHg/sec) (mmHg/sec) (mmHg/min) Control 277 ± 3436 ± 46 12000 ± 1580  7200 ± 408 120,833 ± 13,147 AC + Saline  238 ±12* 334 ± 47 9480 ± 480*  6000 ± 360*  80,860 ± 15,271* AC + TAU & TAC308 ± 9 446 ± 33 11520 ± 600  9600 ± 480 138,056 ± 12,234

[0143] The parameters of myocardial performance were monitored in thesame rats following administration of 0.1 ml of 10⁻⁴ M norepinephrinebitartrate. These values represent the maximal performance of the heart,and are displayed in Table 2 and FIGS. 6-10.

Discussion

[0144] Providing exogenous nucleosides to the myocardium byadministering the acyl nucleoside derivatives of the invention preventsor alleviates the impairments in myocardial performance that normallyaccompany cardiac hyperfunction and hypertrophy that follows a sustainedincrease in load upon the heart. Such an increase in workload occurs inthe surviving portions of the heart following a severe myocardialinfarction. Therefore, pyrimidine nucleosides or acylated derivativesare useful therapeutic agents in the treatment of or prevention of heartfailure following myocardial infarction. There are currently notherapeutic agents in contemporary clinical practice that operate bysupporting the biochemical mechanism underlying myocardial energymetabolism or capacity for adaptation to sustained increases inworkload. These results indicate that such an approach yieldssignificant functional benefits.

Example IV

[0145] Treatment of Liver Damage With Acylated Pyrimidine Nucleosides

[0146] The effect of oral triacetylcytidine and triacetyluridine onchemically induced liver damage was assessed. Chronic treatment ofrodents with carbon tetrachloride is a standard model for inducing aheptatopathy that eventually leads to cirrhosis.

[0147] 20 male F344 rats (200 g) received injections of carbontetrachloride (0.2 ml/kg of 50% CCl₄ in corn oil) twice per week for 8weeks. After the first 2 weeks of treatment with carbon tetrachloride,half of the animals were subjected to oral administration (gavage) of amixture of triacetyluridine (TAU) and triacetylcytidine (TAC) (50 mg/kgof each in 1 ml of water, twice per day) for the remaining 6 weeks. Theother half of the animals (controls) received equivalent volumes ofwater by gavage. At the end of 8 weeks of carbon tetrachloridetreatment, the functional capacity of the livers was assessed by theircapacity to remove bromsulphthalein (BSP) from the circulation (astandard test of liver function). The rats were anesthetized (ketamine80 mg/kg and xylazine 13 mg/kg) and their carotic arteries werecatheterized for BSP administration and blood sampling. BSP (50 mg/kg in0.5 ml saline) was administered as a bolus. Blood samples (0.2 ml) weretaken periodically, and plasma BSP concentrations were determined byadding 20 microliters of plasma to 1 ml 0.1 M NaOH and recording UVabsorbance at 575 nm.

[0148] As is shown in FIG. 11, animals that received TAC and TAU duringtreatment with carbon tetrachloride had a significantly better capacityto remove BSP from their circulation than did the control animals,indicating that TAC and TAu provide significant protection of the liveragainst damage from carbon tetrachloride.

Example V

[0149] Preparation of 2′, 3′, 5′-Tri-O-Acyl Uridine from AcidAnhydrides:

[0150] To 1 gram of uridine dissolved in 20 ml anhydrous pyridine(previously dried over potassium hydroxide) is added at room temperature3.1 molar equivalents of the acid anhydride of the desired acyl compound(e.g., acetic anhydride, lactate anhydride, butyric anhydride, etc.).The reaction mixture is then heated to 80-85° C. for 2 hours, cooled,poured into ice water, and the esters recovered by extraction threetimes with equal volumes of chloroform. The chloroform is then washedwith ice-cold 0.01 N sulfuric acid, 1% aqueous sodium bicarbonate, andfinally water. After drying with sodium sulfate, the chloroform isevaporated and the residual oil or crystals are subjected tochromatography (adapted from Nishizawa et al., Biochem. Pharmacol.14:1605 (1965).

[0151] From acid chlorides:

[0152] To 1 gram of uridine in 20 ml anhydrous pyridine is added, at 5°C., 3.1 molar equivalents of the acid chloride of the desired acylcompound (e.g., palmitoyl chloride, acetyl chloride, etc.). The mixtureis held at room temperature overnight, added to ice water, and worked upas indicated above (adapted from Nishizawa et al., Biochem. Pharmacol.14:1604 (1965)).

Example VI

[0153] Preparation of 5-Acyl Uridine

[0154] To 1 gram of uridine dissolved in 20 ml anhydrous pyridine isadded, at room temperature, 1.0 molar equivalent of the acid anhydrideof the desired acyl compound. The reaction is then heated to 80-85° C.for two hours, cooled, poured into ice water, and the esters recoveredby extraction three times with equal volumes of chloroform. Thechloroform is then washed ice cold 0.01 N sulfuric acid, 1% aqueoussodium bicarbonate, and finally water. After drying with sodium sulfate,the chloroform is evaporated and the residual oil or crystals aresubjected to chromatography. The major product, which is isolated bychromatography, is the 5′-substituted ester (adapted from Nishizawa etal., Biochem. Pharmacol. 14:1605 (1965)).

[0155] Alternatively, selective 5′-acylation of uridine may beaccomplished by suspending 1 gram of uridine in 30 ml of 1:1pyridine:N,N-dimethylformamide cooled to 0° C. in an ice bath. 1.0 molarequivalent of the acid chloride of the desired acyl compound is addeddropwise to the mixture, which is stirred at 0° C. for 12-24 hours. 3 mlof water is added, and then the solvents are evaporated in vacuo at 50°C. The residue is dissolved in methanol and adsorbed onto approximately3 grams of silica gel, and the excess solvent is evaporated off. Tolueneis evaporated three times from the solid mass, and the whole is loadedonto a 3×15 cm slurry-packed column of silica gel in chloroform, andeluted with a linear gradient of chloroform (200 ml) to 20:80methanol:chloroform (200 ml). The appropriate fractions, as determinedby TLC, are combined, and the solvents are evaporated to yield thedesired product that is either recrystallized or dried in vacuo to aglass (adapted from Baker et al., J. Med. Chem. 21:1218 (1978)).

Example VII

[0156] Preparation of 2′, 3′-Diacyl Uridine

[0157] To a stirred suspension of 1 gram of uridine in 20 ml dry N,N-dimethylformamide is added 2.4 molar equivalents of imidazole followedby 1.2 molar equivalents of t-butyldimethylchlorosilane. The mixture isstirred with protection from moisture at room temperature for 20 hours,at which time the solvent is removed at 50° C. in vacuo. The residue isdissolved in 15 ml of ethyl acetate, the solution is washed with 10 mlof water, and the extract is dried with magnesium sulfate and evaporatedto give a syrup. Crystallization from 10 ml of hot chloroform, to whichis added hexane to the point of opalescence, followed by slow cooling toroom temperature, gives 5′-(t-butyldimethylsilyl) uridine.

[0158] To a stirred suspension of 1 gram of 5′-(t-butyldimethylsilyl)uridine in 15 ml of dry pyridine cooled to 0° C., is added 2.1 molarequivalents of the appropriate acid anhydride of the desired acylcompound, and the mixture is stirred with protection from moisture for20 hours at 0-5° C., at which time the reaction is terminates byaddition of a few ml of water. The solvent is evaporated and the residueis dissolved in 15 ml of chloroform, washed with 2×15 ml of saturatedsodium hydrogen carbonate, and then with water, dried (magnesiumsulfate) and evaporated to give a thick, clear syrup, which is thendried in vacuo at 25° C.

[0159] To a stirred solution of the above acylated Product in 30 ml ofdry tetrahydrofuran is added 0.2 ml glacial acetic acid, followed by1.5-2.3 grams of tetrabutylammonium fluoride, and the reaction ismonitored by TLC (9:1 chloroform methanol). Upon complete removal of thet-butyldimethylsilyl group from the 5′ hydroxyl group or the acylateduridine derivative, the fluoride is removed from the mixture byfiltration through a layer of 30 grams of silica gel, and the productsare eluted with tetrahydrofuran. The crude product, obtained uponevaporation of the solvent is recrystallized from acetone, yielding thedesired 2′3′-diacyl uridine derivative (adapted from Baker et al., J.Med. Chem. 22:273 (1979)).

Example VIII

[0160] Preparation of N³, 2′, 3, 5′Tetraacyl Uridine

[0161] The acylation of the secondary amine in the 3 position of thepyrimidine ring is accomplished by reacting 2′, 3′, 5′-tri-O-acyluridine with 1.1 molar equivalents of the acid chloride of the desiredacyl substituent in an aprotic solvent (such as ether, dioxane,chloroform, ethyl acetate, acetonitrile, pyridine, dimethylformamide andthe like) in the presence of 1-5 molar equivalents of an organic base(especially aromatic amines such as pyridine, trialkylamines, orN,N-dialkylanilines) adapted from Fujii et al., U.S. Pat. No.4,425,335). The acyl substituent on the secondary amine can be the sameor different from those on the hydroxyl groups of the ribose moiety.

Example IX

[0162] Preparation of 2′, 3′, 5′-Tri-O-acyl Cytidine

[0163] One gram of cytidine hydrochloride is dissolved in 10 ml ofN,N-dimethylformamide. 3.1 molar equivalents of the acid chloride isadded and the mixture is stirred overnight at room temperature. Thereaction mixture is concentrated in vacuo to an oil, and triturated with1.1 ethyl acetate: diethyl ester. The oil is then triturated with 1Nsodium hydrogen carbonate. The crystalline solid is collected, washedwith water, dried, and recrystallized (adapted from Gish et al., J. Med.Chem.14:1159 (1971)).

Example X

[0164] Preparation of 5′-Acyl Cytidine

[0165] One gram of cytidine hydrochloride is dissolved in 10 ml ofN,N-dimethylformamide. 1.1 molar equivalents of the acid chloride of thedesired acyl substituent is added, and the mixture is stirred overnightat room temperature. The reaction mixture is concentrated in vacuo to anoil, and triturated with 1:1 ethyl acetate: diethyl ether. The oil isthen triturated with 1N sodium hydrogen carbonate. The crystalline solidis collected, washed with water, dried, and recrystallized (adapted fromGish et al., J. Med. Chem. 14:1159 (1971)).

Example XI

[0166] Preparation of N⁴-Acyl Cytidine

[0167] The N⁴-amino group of cytidine is the best nucleophile among theamino and hydroxyl functionalities of cytidine. Selective N⁴-acylationcan be accomplished by treating cytidine with appropriate acidanhydrides in pyridine or a mixture of pyridine andN,N-dimethylformamide. Specifically, 1 gram of cytidine is suspended in80 ml of dry pyridine; 1.5 molar equivalents of desired acid anhydrideis added, and the mixture is refluxed for 2 hours. The solvent isremoved in vacuo, and the resulting white solid is recrystallized fromethanol.

[0168] Alternatively, cytidine (1 gram) is dissolved in a mixturecomprising 70:30 pyridine:N,N-dimethylformamide. 1.5 molar equivalentsof the acid anhydride of the desired acyl substituent is added, and themixture is stirred overnight at room temperature, after which it ispoured into water and stirred. The solvent is removed in vacuo to leavea white solid, which is extracted with diethy ether. The residue isrecrystallized from ethanol (adapted from Sasaki et al., Chem. Pharm.Bull 15:894 (1967)).

[0169] An alternative procedure is to dissolve cytidine in a mixture ofwater and a water-miscible organic solvent (such as dioxane, acetone,acetonitrile, N,N-dimethylformamide, dimethylsulfoxide, tetrahydrofuran,etc.) and to treat that solution with about a twofold excess of anappropriate acid anhydride. For example, 1 gram of cytidine dissolved in5 ml of water is mixed with 15 to 100 ml dioxane (more dioxane is neededfor more lipophilic substituents), and 2 molar equivalents of the acidanhydride of the desired acyl substituent is added. The mixture isstirred for 5 hours at 80° C. (or 48 hours at room temperature), andthen the solvent is removed in vacuo. The residue is washed with hexaneor benzene, and recrystallized from ethanol or ethyl acetate (adaptedfrom Akiyama et al., Chem. Pharm. Bull. 26:981 (1978).

Example XII

[0170] Preparation of N⁴, 2′, 3′, 5′Tetraacyl Cytidine

[0171] Compounds in which the acyl substituent of the N⁴ amino group andthe hydroxyl groups of the ribose ring of cytidine are the same (e.g.,tetraacetyl cytidine) are prepared by dissolving or suspending cytidinein dry pyridine, adding at least 4 molar equivalents of the acidchloride or acid anhydride of the desired substituent, and stirring themixture overnight at room temperature. The solvent is removed in vacuoand the residue is washed and recrystallized.

[0172] Having now fully described this invention, it will be appreciatedby those skilled in the art that the same can be performed within a widerange of equivalent parameters of composition, conditions, and modes ofadministration without departing from the spirit or scope of theinvention or any embodiment thereof.

What is claimed is:
 1. An acyl derivative of uridine having the formula(II)

wherein R₁, R₂, and R₃ are the same or different and each is hydrogen oran acyl radical of (a) an unbranched fatty acid with 5 to 22 carbonatoms, (b) an amino acid selected from the group consisting of glycine,L-forms of alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cystine, cysteine, aspartic acid,glutamic acid, arginine, lysine, histidine, carnitine, and ornithine,(c) a dicarboxylic acid of 3 to 22 carbon atoms, or (d) a carboxylicacid selected from one or more of the group consisting of glycolic acid,pyruvic acid, lactic acid, enolpyruvic acid, lipoic acid, pantothenicacid, acetoacetic acid, p-aminobenzoic acid, betahydroxybutyric acid,orotic acid, and creatine, provided that at least one of saidsubstituents R₁, R₂, and R₃ is not hydrogen, and further provided thatif any of said substituents R₁, R₂, and R₃ is hydrogen and if saidremaining substituents are acyl radicals of a straight chain fatty acid,then said straight chain fatty acid has 8 to 22 carbon atoms, or apharmaceutically acceptable salt thereof.
 2. An acyl derivative ofuridine having the formula (I)

wherein R₁, R₂, and R₃ are the same or different and each is hydrogen oran acyl radical of a metabolite, and R₄ is an acyl radical of ametabolite, or a pharmaceutically acceptable salt thereof.
 3. An acylderivative of uridine as recited in claim 2 wherein said metabolite isan acyl radical of a carboxylic acid selected from one or more of thegroup consisting of a fatty acid of 2 to 22 carbon atoms, glycolic acid,pyruvic acid, lactic acid, enolpyruvic acid, an amino acid, lipoic acid,pantothenic acid, succinic acid, fumaric acid, adipic acid, acetoaceticacid, p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, andcreatine, or a pharmaceutically acceptable salt thereof.
 4. The acylderivative of claim 3 wherein said amino acid is selected from the groupconsisting of glycine, the L forms of alanine, valine, leucine,isoleucine, proline, phenylalanine, tyrosine, cysteine, cystine,methionine, tryptophan, aspartic acid, glutamic acid, arginine, lysine,histidine, ornithine, carnitine, and hydroxylysine.
 5. A compositioncomprising the acyl derivative of claims 1 or 2 and a pharmaceuticallyacceptable carrier.
 6. A unit dose of the composition of claim 5comprising an amount of said acyl derivative being the equivalent of10-3000 mg of uridine.
 7. A composition comprising a mixture of at leastone acyl derivative of claims 1 or 2, at least one acyl derivative ofcytidine selected from the group consisting of 2′, 3′, 5′-tri-O-acetylcytidine, 2′, 3′, 5′-tri-O-propionyl cytidine, or 2′, 3′,5′-tri-O-butyryl cytidine and a pharmaceutically acceptable carrier. 8.A unit dose of the composition of claim 7 comprising amounts of saidacyl derivatives being the equivalent of 10-3000 mg of uridine and10-3000 mg of cytidine.
 9. The composition of claim 5 or 7 in the formof a liquid, a suspension, a tablet, a dragee, an injectable solution,or a suppository.
 10. A method of delivering exogenous uridine to thetissue of an animal, comprising the step of administering to said animalan effective amount of an acyl derivative of uridine as recited in claim1 or
 2. 11. A method of delivering exogenous uridine to the tissue of ananimal, comprising the step of administering to said animal an effectiveamount of an acyl derivative of uridine having the formula (I)

wherein R₁, R₂, R₃, and R₄ are the same or different and each ishydrogen or an acyl radical of a metabolite, provided that at least oneof said R substituents is not hydrogen, or a pharmaceutically acceptablesalt thereof.
 12. A method as recited in claim 11 wherein saidmetabolite is a carboxylic acid selected from one or more of the groupconsisting of glycolic acid, pyruvic acid, lactic acid, enolpyruvicacid, an amino acid, a fatty acid of 2 to 22 carbon atoms, lipoic acid,pantothenic acid, succinic acid, fumaric acid, adipic acid, acetoaceticacid, p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, andcreatine.
 13. A method of delivering exogenous cytidine to the tissue ofan animal, comprising the step of administering to said animal aneffective amount of an acyl derivative of cytidine having the formula(III)

wherein R₁, R₂, R₃, and R₄ are the same or different and each ishydrogen or an acyl radical of a metabolite provided that at least oneof said R substituents is not hydrogen, or a pharmaceutically acceptablesalt thereof.
 14. A method as recited in claim 13 wherein saidmetabolite is a carboxylic acid selected from one or more of the groupconsisting of glycolic acid, pyruvic acid, lactic acid, enolpyruvicacid, an amino acid, a fatty acid of 2 to 22 carbon atoms, lipoic acid,pantothenic acid, succinic acid, fumaric acid, adipic acid, acetoaceticacid, p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, andcreatine.
 15. A method of treating physiological or pathologicalconditions of the tissue of an animal by supporting metabolic functionsthereof, comprising increasing the bioavailability of uridine to saidtissue by administering to said animal an effective amount of an acylderivative of uridine as recited in claim 1 or
 2. 16. A method oftreating physiological or pathological conditions of the tissue of ananimal by supporting metabolic functions thereof, comprising increasingthe bioavailability of uridine to said tissue by administering to saidanimal an effective amount of an acyl derivative of uridine having theformula (I)

wherein R₁, R₂, R₃, and R₄ are the same or different and each ishydrogen or an acyl radical of a metabolite, provided that at least oneof said R substituents is not hydrogen, or a pharmaceutically acceptablesalt thereof.
 17. A method as recited in claim 16 wherein saidmetabolite is a carboxylic acid selected from one or more of the groupconsisting of acetic acid, glycolic acid, pyruvic acid, lactic acid,enolpyruvic acid, an amino acid, a fatty acid of 2 to 22 carbon atoms,lipoic acid, pantothenic acid, succinic acid, fumaric acid, adipic acid,acetoacetic acid, p-aminobenzoic acid, betahydroxybutyric acid, oroticacid, and creatine.
 18. A method of treating physiological orpathological conditions of the tissue of an animal by supportingmetabolic functions thereof, comprising increasing the bioavailabilityof cytidine to said tissue by administering to said animal an effectiveamount of an acyl derivative of cytidine having the formula (III)

wherein R₁, R₂, R₃, and R₄ are the same or different and each ishydrogen or an acyl radical of a metabolite, provided that at least oneof said R substituents is not hydrogen, or a pharmaceutically acceptablesalt thereof.
 19. A method as recited in claim 18 wherein saidmetabolite is a carboxylic acid selected from one or more of the groupconsisting of glycolic acid, pyruvic acid, lactic acid, enolpyruvicacid, an amino acid, a fatty acid of 2 to 22 carbon atoms, lipoic acid,pantothenic acid, succinic acid, fumaric acid, adipic acid, acetoaceticacid, p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, andcreatine.
 20. A composition comprising a mixture of at least one acylderivative of uridine as recited in claims 1, 2, or 16, at least oneacyl derivative of cytidine as recited in claim 20 and apharmaceutically acceptable carrier.
 21. A method for treating cardiacinsufficiency, myocardial infarction, hepatopathy, diabetes,cerebrovascular disorders Parkinson's disease, or to enhance muscleperformance, or to improve immune responses, comprising administering toan animal an effective amount of a composition comprising an acylderivative of uridine having the formula (I)

wherein R₁, R₂, R₃ and R₄ are the same or different and each is hydrogenor an acyl radical of a metabolite, provided that at least one of said Rsubstituents is not hydrogen, or a pharmaceutically acceptable saltthereof.
 22. A method for treating cardiac insufficiency, myocardialinfarction, hepatopathy, diabetes, cerebrovascular disorders,Parkinson's disease, and infant respiratory distress syndrome, or toenhance muscle performance, or to improve immune responses comprisingadministering to an animal an effective amount of a compositioncomprising an acyl derivative of cytidine, having the formula (III)

wherein R₁, R₂, R₃, and R₄ are the same or different and each is an acylradical of a metabolite, provided that at least one of said Rsubstituents is not hydrogen, or a pharmaceutically acceptable saltthereof.
 23. A method for treating cardiac insufficiency, myocardialinfarction, hepatopathy, diabetes, cerebrovascular disorders,Parkinson's disease, or to enhance muscle performance, or to improveimmune responses, comprising coadministering an effective amount of atleast one acyl derivative of uridine as recited in claim 21 and at leastone acyl derivative of cytidine as recited in claim
 22. 24. The methodof claim 23, wherein said coadministered acyl derivatives comprise atleast one derivative selected from the group consisting of 2′, 3′,5′-tri-O-acetyl cytidine, 2′, 3′, 5′-tri-O-propionyl cytidine, and 2′,3′, 5′-tri-O-butyryl cytidine, and at least one derivative selected fromthe group consisting of 2′, 3′, 5′-tri-O-acetyl uridine, 2′, 3′,5′-tri-O-propionyl uridine, and 2′, 3′, 5′-tri-O-butyryl uridine. 25.The method of claim 24, wherein the dose of each of said uridinederivatives is 15-4500 mg and the dose of each of said cytidinederivatives is 15-4500 mg.
 26. A method as recited in claim 11 whereinsaid exogenous uridine is delivered from the gastrointestinal tract intothe circulation.
 27. A method as recited in claim 13 wherein saidexogenous cytidine is delivered from the gastrointestinal tract into thecirculation.
 28. A method as recited in claim 26 wherein an effectiveamount of 2′, 3′, 5′-tri-O-acetyl uridine, 2′, 3′, 5′-tri-O-propionyluridine, or 2′, 3′, 5′-tri-O-butyryl uridine, or pharmaceuticallyacceptable salts thereof is administered to said animal.
 29. A method asrecited in claim 27 wherein an effective amount of 2′, 3′,5′-tri-O-acetyl cytidine, 2′, 3′, 5′-tri-O-propionyl cytidine, or 2′,3′, 5′-tri-O-butyryl cytidine, or pharmaceutically acceptable saltsthereof is administered to said animal.
 30. A composition for deliveringexogenous uridine to the tissue of an animal comprising an effectiveamount of an acyl derivative of uridine as recited in claim 11 and apharmaceutically acceptable carrier.
 31. A composition for deliveringexogenous cytidine to the tissue of an animal comprising an effectiveamount of an acyl derivative of cytidine as recited in claim 13 and apharmaceutically acceptable carrier.
 32. A composition for treatingphysiological or pathological conditions of the tissue of an animal bysupporting metabolic functions thereof comprising an effective amount ofan acyl derivative of uridine as recited in claim 16 and apharmaceutically acceptable carrier.
 33. A composition for treatingphysiological or pathological conditions of the tissue of an animal bysupporting metabolic functions thereof comprising an effective amount ofan acyl derivative of cytidine as recited in claim 18 and apharmaceutically acceptable carrier.
 34. A composition for treatingphysiological or pathological conditions of the tissue of an animal bysupporting metabolic functions thereof comprising an effective amount ofat least one acyl derivative as recited in claim 16 and at least oneacyl derivative of cytidine as recited in claim 18, and apharmaceutically acceptable carrier.
 35. A composition as recited inclaim 30 wherein said acyl derivative of uridine is 2′, 3′,5′-tri-O-acetyl uridine, 2′, 3′, 5′-tri-O-propionyl uridine, or 2′, 3′,5′-tri-O-butyryl uridine.
 36. A composition as recited in claim 31wherein said acyl derivative of cytidine is 2′, 3′, 5′-tri-O-acetylcytidine, 2′, 3′, 5′-tri-O-propionyl cytidine, or 2′, 3′,5′-tri-O-butyryl cytidine.
 37. A composition as recited in claim 32wherein said acyl derivative of uridine is 2′, 3′, 5′-tri-O-acetyluridine, 2′, 3′, 5′-tri-O-propionyl uridine, or 2′, 3′, 5′-tri-O-butyryluridine.
 38. A composition as recited in claim 33 wherein said acylderivative of cytidine is 2′, 3′, 5′-tri-O-acetyl cytidine, 2′, 3′,5′-tri-O-propionyl cytidine, and 2′, 3′, 5′-tri-O-butyryl cytidine. 39.A composition as recited in claim 34 wherein said acyl derivative ofuridine is selected from the group consisting of 2′, 3′, 5′-tri-O-acetyluridine, 2′, 3′, 5′-tri-O-propionyl uridine, and 2′, 3′,5′-tri-O-butyryl uridine, and said acyl derivative of cytidine isselected from the group consisting of 2′, 3′, 5′-tri-O-acetyl cytidine,2′, 3′, 5′-tri-O-butyryl cytidine.